The ability of the estrogen receptor (ER) to function as a ligand- mediated transcription factor requires the participation of residues from multiple regions throughout the ligand binding domain (LBD). These regions collaborate with the remainder of the receptor to mediate a wide spectrum of activities observed in response to physiologic and synthetic ligands which are varied in their structures. An understanding of the capacity of ER to accommodate such a wide variety of ligand structures and the gene selective control which results from the complexes formed, will be the focus of these investigations. Progress in this laboratory has shown that A-ring isomers of estradiol (E2) can induce dramatic changes in the activation profiles of certain ER-regulated genes. Mutant ERs with substitutions to residues in the AF2 activation function demonstrate tremendous sensitivity to alterations in the structure of the binding ligand. Point mutations to residues on the polar side of the AF2 helix produce ERs which become activated in response to stimulation with inert isomers of E2. Agonism can be induced in response to structurally diverse antagonists when bound to ER mutants bearing changes to residues on the non polar side of the AF2 helix. The goal of this proposal is to examine the mechanism used by the ER to distinguish between different elements of ligand structure and to discern the receptor residues involved in the transfer of this information to regulatory interaction surfaces on the ER which maybe required for coactivation. These investigations will use as probing ligands the A-ring isomers of E2, monohydroxyestrogens, and certain other estrogen analogs to trace the path of ligand-induced activation in the LBD of the ER protein. Specifically, the proposed experiments will employ the design and use of selected mutants of ER to examine the effects that structurally altered estrogens have on transactivation and coactivator-mediated enhancement. This will be accomplished using both the alpha and beta forms of ER and it will include an assessment of conformational change and phenotypic dominance. Results from these studies are expected to advance our understanding of the ligand-based transcriptional control mechanisms which are used by the ER and to produce information which can aid in the design of new ligands to control estrogen activity in hormone dependent breast cancer, osteoporosis, endometrial cancer, and fertility.